Bottom Line:
Holtgreve-Grez, A.Curr.These results demonstrate that the timely removal of sister telomere cohesion is essential for the formation of a protective structure at chromosome ends after DNA replication in S/G2 phase of the cell cycle.

Affiliation: Molecular Pathogenesis Program, The Helen L. and Martin S. Kimmel Center for Biology and Medicine, Skirball Institute of Biomolecular Medicine, New York University School of Medicine, New York, NY 10016, USA.

ABSTRACTTelomeres protect chromosome ends from being viewed as double-strand breaks and from eliciting a DNA damage response. Deprotection of chromosome ends occurs when telomeres become critically short because of replicative attrition or inhibition of TRF2. In this study, we report a novel form of deprotection that occurs exclusively after DNA replication in S/G2 phase of the cell cycle. In cells deficient in the telomeric poly(adenosine diphosphate ribose) polymerase tankyrase 1, sister telomere resolution is blocked. Unexpectedly, cohered sister telomeres become deprotected and are inappropriately fused. In contrast to telomeres rendered dysfunctional by TRF2, which engage in chromatid fusions predominantly between chromatids from different chromosomes (Bailey, S.M., M.N. Cornforth, A. Kurimasa, D.J. Chen, and E.H. Goodwin. 2001. Science. 293:2462-2465; Smogorzewska, A., J. Karlseder, H. Holtgreve-Grez, A. Jauch, and T. de Lange. 2002. Curr. Biol. 12:1635-1644), telomeres rendered dysfunctional by tankyrase 1 engage in chromatid fusions almost exclusively between sister chromatids. We show that cohered sister telomeres are fused by DNA ligase IV-mediated nonhomologous end joining. These results demonstrate that the timely removal of sister telomere cohesion is essential for the formation of a protective structure at chromosome ends after DNA replication in S/G2 phase of the cell cycle.

fig3: Tankyrase 1 depletion leads to persistent sister telomere cohesion in HTC75, BJ, and BJ + TERT cells. (A–F) Visualization and quantification of chromosome-specific FISH analysis of HTC75 (A and B), BJ (C and D), or BJ + TERT (E and F) cells after infection with GFP or TNKS1 lentiviral shRNA. At 48 h after infection, colcemid was added for 12 h, and cells were collected by mitotic shake-off. Cells were fixed directly with methanol/acetic acid without hypotonic swelling and hybridized to a subtelomeric 16pter probe (green). DNA is stained with DAPI (blue). (B, D, and F) Quantification of mitotic cells with unseparated telomeres. Approximately 100 mitotic cells were scored for each sample. Bar, 2 µm.

Mentions:
As described in the previous section, depletion of tankyrase 1 in three different human cell lines (HTC75, BJ, and BJ + TERT) led to a DNA damage response and a senescence-like G1 arrest. This cellular response was dramatically different from the mitotic arrest observed upon tankyrase 1 depletion in HeLa cells (Dynek and Smith, 2004; Chang et al., 2005). Therefore, we wondered whether (despite the different phenotypes observed) tankyrase 1 depletion was still causing the same telomere dysfunction in the aforementioned HTC75 and BJ cell lines. To address this question, HTC75 cells were infected with tankyrase 1 or GFP shRNA lentivirus. Cells were isolated 48 h after infection by mitotic shake-off and analyzed by chromosome-specific telomere FISH using a 16pter subtelomere probe. As shown in Fig. 3 A, in control mitotic cells (GFP shRNA), sister telomeres appear as doublets, indicating normal resolution of sister telomere cohesion. In contrast, in tankyrase 1–depleted cells (TNKS1 shRNA), sister telomeres appear as singlets, indicating a block in resolution of sister telomere cohesion. Quantification of this analysis shows a >10-fold increase in unresolved sister telomeres (Fig. 3 B). Thus, tankyrase 1–depleted HTC75 cells, like HeLa cells (Dynek and Smith, 2004; Canudas et al., 2007), show a block in resolution of sister telomere cohesion. Similar results were obtained for BJ (Fig. 3, C and D) and BJ + TERT (Fig. 3, E and F) cells.

Bottom Line:
Holtgreve-Grez, A.Curr.These results demonstrate that the timely removal of sister telomere cohesion is essential for the formation of a protective structure at chromosome ends after DNA replication in S/G2 phase of the cell cycle.

Affiliation:
Molecular Pathogenesis Program, The Helen L. and Martin S. Kimmel Center for Biology and Medicine, Skirball Institute of Biomolecular Medicine, New York University School of Medicine, New York, NY 10016, USA.

ABSTRACTTelomeres protect chromosome ends from being viewed as double-strand breaks and from eliciting a DNA damage response. Deprotection of chromosome ends occurs when telomeres become critically short because of replicative attrition or inhibition of TRF2. In this study, we report a novel form of deprotection that occurs exclusively after DNA replication in S/G2 phase of the cell cycle. In cells deficient in the telomeric poly(adenosine diphosphate ribose) polymerase tankyrase 1, sister telomere resolution is blocked. Unexpectedly, cohered sister telomeres become deprotected and are inappropriately fused. In contrast to telomeres rendered dysfunctional by TRF2, which engage in chromatid fusions predominantly between chromatids from different chromosomes (Bailey, S.M., M.N. Cornforth, A. Kurimasa, D.J. Chen, and E.H. Goodwin. 2001. Science. 293:2462-2465; Smogorzewska, A., J. Karlseder, H. Holtgreve-Grez, A. Jauch, and T. de Lange. 2002. Curr. Biol. 12:1635-1644), telomeres rendered dysfunctional by tankyrase 1 engage in chromatid fusions almost exclusively between sister chromatids. We show that cohered sister telomeres are fused by DNA ligase IV-mediated nonhomologous end joining. These results demonstrate that the timely removal of sister telomere cohesion is essential for the formation of a protective structure at chromosome ends after DNA replication in S/G2 phase of the cell cycle.